Antifibrotic therapy

ABSTRACT

The present invention relates to methods of treating severe or rapidly progressing pulmonary fibrosis in a subject in need of a treatment thereof. The methods comprise increasing the activity of pulmonary and activation-regulated chemokine (CCL18) in the lungs of the subject, whereby increasing CCL18 activity modulates the activity of at least one antifibrotic factor in the lungs of the subject. The present invention also relates to methods of screening test procedures that may be capable of treating severe or rapidly progressing pulmonary fibrosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/989,076, filed 19 Nov. 2007, which is incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research performed during development of this invention utilized U.S.Government funds from NIH Grant Number HL074067 and VA Merit Review TypeI Award. The U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of treating severe or rapidlyprogressing pulmonary fibrosis in a subject in need of a treatmentthereof. The methods comprise increasing the activity of pulmonary andactivation-regulated chemokine (CCL18) in the lungs of the subject;whereby increasing CCL18 activity modulates the activity of at least oneantifibrotic factor in the lungs of the subject.

In particular, the present invention relates to methods of treating,preventing, or preventing the progression of pulmonary fibrosis.

2. Background of the Invention

Pulmonary fibrosis is usually diagnosed by a history of progressiveshortness of breath with exertion, crackling sounds in the chest bystethoscope examination, abnormal CAT scan, and/or abnormal lungfunctioning test. The diagnosis may also be confirmed by lung biopsy inwhich the chest wall is surgically opened under general anesthesia toremove a portion of lung tissue, and the removed tissue is examinedmicroscopically to confirm the presence of fibrosis. Schiffman G.,Pulmonary Fibrosis, Medicinenet.com (Stoppler M.C., ed., last editorialreview Dec. 6, 2006).

The treatment for idiopathic pulmonary fibrosis is very limited, andthere is presently no evidence that any medication(s) can treat thiscondition, since scarring is usually permanent throughout the lungs.Currently, lung transplantation is the only therapeutic optionavailable. Studies exploring drug treatment to reduce fibrous scarringare ongoing. For example, because the immune system is centrallyimplicated in the development of pulmonary fibrosis, corticosteroids(e.g., prednisone) is used to suppress the immune system in order toattenuate physiological processes that lead to fibrosis, e.g., todecrease lung inflammation and subsequent scarring. However, the sideeffects and toxicity of drug treatments may be serious. In this regard,only a minority of patients respond to corticosteroids alone, soadditional immunosuppressive medications are used in conjunction withcorticosteroids. These drugs include gamma-interferon, cyclophosphamide,azathioprine, methotrexate, penicillamine, cyclosporine, andanti-inflammatory medications, but have met with limited success.

Patients with pulmonary fibrosis are subjected to a vicious cycle ofoxygen deprivation that requires supplemental oxygen treatment toprevent pulmonary hypertension. This type of hypertension is caused byan initial decrease in blood oxygen levels that creates a hypoxicsituation that leads to the pulmonary hypertension from the elevatedpressure in the pulmonary artery. This pulmonary hypertension eventuallyleads to failure of the right ventricle of the heart. Schiffman G.,Pulmonary Fibrosis, Medicinenet.com (Stoppler M.C., ed., last editorialreview Dec. 6, 2006).

The present inventors have recently reported that levels of a CCchemokine CCL18 mRNA and protein are increased in alveolar macrophagesand bronchoalveolar lavage (BAL) fluid, respectively, in patients withscleroderma lung disease (1), a condition characterized by theaccumulation of T cells in the lungs and by pulmonary fibrosis (2).Increases in CCL18 have also been reported in the lungs of patients withother pulmonary diseases characterized by T cell involvement andcollagen deposition, such as hypersensitivity pneumonitis and idiopathicpulmonary fibrosis (3,4), pulmonary sarcoidosis (5), and allergic asthma(6,7).

CCL18, also known as pulmonary and activation-regulated chemokine(PARC), macrophage inflammatory protein 4 (MIP-4), alternativemacrophage activation-associated CC chemokine 1 (AMAC-1), dendriticcell-derived chemokine1 (DCCK1), and small secreted cytokine A 18(SCYA-18), is constitutively expressed at high levels in the lungs(3,8-10) and is selectively chemotactic for T cells (11).

The present inventors have found (12-14), and others have recentlyconfirmed (4) that CCL18 in high concentrations (300-1000 ng/ml) actsdirectly on cultured primary pulmonary fibroblasts, activatesintracellular signaling, and stimulates collagen production in a time-and dose-dependent manner.

Macrophages produce CCL18 alternative activation in a Th2 environment(8,15-17) and directly stimulate collagen production in fibroblasts(18). In vivo, CCL18 may promote pulmonary fibrosis when expressed atmuch lower concentrations (300 pg/m), by attracting T cells to the lungs(11).

T cells constitute a relatively minor population in a normal lung,although the T cell population expands numerically and undergoesphenotypic changes in association with lung inflammation and fibrosis(2,19-22). Although not all pulmonary fibrotic processes are T celllymphocyte-dependent (23-25), previous studies suggest that Tlymphocytes contribute to regulation of fibrosis in the lung in humandisease (1,2,19-21) and in animal models of pulmonary fibrosis(22,26,27).

In contrast to pulmonary T lymphocytes, macrophages are by far the mostabundant cell type in the lungs, normally constituting more than 85% ofbronchoalveolar lavage cells (2). Although the percentage of macrophagesdeclines during lung inflammation due to influx of T cells and otherinflammatory cells, macrophages undergo phenotypic changes associatedwith inflammation and fibrosis (1), and establish a vicious circle ofpulmonary fibrosis by further upregulating CCL18 expression (4).

The present inventors have developed a CCL18 overexpression animal modelthat resembles human pulmonary fibrotic disease. The animal modelmanifests pulmonary T lymphocytic infiltration, TGF-βactivation, and Tcell-dependent collagen accumulation (11). However, in humans withpulmonary fibrosis, the elevation of pulmonary levels of CCL18 occurs inthe context of pulmonary inflammation and fibrosis that may affect theoutcome of CCL18 expression in the lungs. Elevated pulmonary levels ofCCL18 have been associated with influx of T lymphocytes, collagenaccumulation, and a decline in lung function in pulmonary fibrosispatients. It was previously reported that overexpression of CCL18 inmouse lungs triggers selective infiltration of T lymphocytes andmoderate lymphocyte-dependent collagen accumulation. Considering thatboth CCL18 expression (11) and inflammation (28) are profibrotic, it washypothesized that the combined action of CCL18 overexpression andbleomycin-induced lung injury would cause a profound, additive orsynergistic, fibrotic lung damage.

The present inventors report here that unexpectedly, increasing CCL18activity in the lungs of an animal with severe or rapidly progressingpulmonary fibrosis is actually protective against chemical-inducedinjured. The mechanisms of this phenomenon are addressed by followingthe changes in the levels of the factors that known to be involved inthe regulation of fibrosis, including matrix metalloproteinases MMP2 andMMP9 and cytokines TGF-β, IL-13, TNF-α, and IFN-γ (29-33).

The present inventors have also found that the CCL8-attracted pulmonaryT cell lymphocytes act profibrotically in otherwise healthy lungs butpartially antifibrotically in the presence of a profibrotic injury(induced by a drug such as bleomycin). The implication of thisobservation is that a therapeutic elimination of T cell lymphocytes fromthe inflamed lungs may have a counterintuitive, deleterious effect inthe patient. Furthermore, there may be potential for further enhancingthe antifibrotic regulation in the lungs by therapeutically manipulatingthe local pulmonary milieu and/or the phenotypes of infiltrating Tlymphocytes.

The present inventors infected mice with a replication-deficientadenovirus encoding CCL18 and instilled a drug that induces pulmonaryinjury (e.g., bleomycin). In addition, control mice were challenged witheither CCL18 overexpression or at least one drug that induces pulmonaryinjury such as inflammation. The additive effects of CCL18overexpression and drug-induced injury were observed on pulmonaryinflammation, particularly on T cell infiltration, and increased levelsof TNF-α, IFN-γ, MMP2, and MMP9.

Despite the hypothesis that CCL18 would have additive effect oninflammation, the present inventors found that CCL18 overexpressionunexpectedly attenuated the drug-induced collagen accumulation.Pulmonary levels of active TGF-β mirrored the changes in collagenlevels. Depletion of T cells with anti-lymphocyte serum orpharmacological inhibition of MMPs with GM6001 abrogated accumulation ofcollagen and increases in the levels of TNF-α, IFN-γ, and active TGF-β.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating severe or rapidlyprogressing pulmonary fibrosis in a subject in need of a treatmentthereof. The methods comprise increasing the activity of pulmonary andactivation-regulated chemokine (CCL18) in the lungs of the subject,whereby increasing CCL18 activity modulates the activity of at least oneantifibrotic factor in the lungs of the subject.

The present invention also relates to methods of treating severe orrapidly progressing pulmonary fibrosis comprising administering a meansfor increasing the activity of pulmonary and activation-regulatedchemokine (CCL18) in the lungs of the subject, whereby increasing CCL18activity modulates the activity of at least one antifibrotic factor inthe lungs of the subject. The means for increasing the activity of CCL18include, but are not limited to, those methods described herein, such astransfection, increasing mRNA stability, increasing the biologicalhalf-life of a peptide and the like.

The present invention also relates to methods of screening a compoundthat may alter the progression of severe or rapidly progressingpulmonary fibrosis. The screening methods comprise administering aninjury-inducing agent to a control and test population of cells, whereinthe injury-inducing agent is known to produce severe or rapidlyprogressing pulmonary fibrosis. A test procedure is also administered tothe test population of injured cells, wherein the test procedure isknown or suspected of being able to increase the activity of pulmonaryand activation-regulated chemokine (CCL18) in cell populations. Data isobserved or gathered wherein the data comprises test level activities ofat least one antifibrotic factor in or from the test population ofcells. The test data are compared to control data wherein the controldata comprises standard activity level of the at least one antifibroticfactor are established in the control population of injured cells. Adifference in test activity levels and standard activity levelsindicates that the test procedure may be capable of altering theprogression of severe or rapidly progressing pulmonary fibrosis.

In one embodiment, the methods of the present invention increase CCL18expression by gene delivery into tar-et cells.

In one embodiment, the target cells of the present invention arepulmonary cells.

In one embodiment of the methods of the present invention, the genedelivery comprises the use of a viral vector. The viral vector maycomprise a replication-deficient recombinant adenoviral vector orreplication-deficient recombinant adeno-associated viral vector.

In another embodiment of the methods of the present invention, the genedelivery comprises the use of a non-viral vector.

In one embodiment, the methods of the present invention increase CCL18expression with viral or non-viral CCL18 gene delivery into targetcells. In other embodiments, the methods of the present inventionincrease CCL18 expression gene delivery comprising magnetofection,cationic lipid-based delivery, electroporation, and a combinationthereof.

In one embodiment, the antifibrotic factors include, but are not limitedto matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9),tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), interleukin-1(IL-1), T cells, B cells, natural killer (NK) cells, interferon gamma(IFN-γ), interferon alpha (IFN-α), and combinations thereof.

In another embodiment, increasing CCL18 expression of the presentinvention modulates the expression of at least one antifibrotic factorselected from the group consisting of MMP2, MMP9, TNF-α, IL-8, IL-1, Tcells, B cells, natural killer (NK) cells, IFN-γ, IFN-α, and acombination thereof.

In one embodiment of the methods of the present invention, the severeand/or rapidly progressing fibrosis is mediated by at least one immunecell selected from the group consisting of monocytes, macrophages,lymphocytes, plasma cells, and a combination thereof.

In one embodiment of the methods of the present invention, the immunecells are macrophages.

In another embodiment of the methods of the present invention, theimmune cells are lymphocytes.

In one embodiment of the methods of the present invention, themacrophages produce cytokines selected from the group consisting ofTNF-α, IL-8, IL-1, and a combination thereof.

In another embodiment of the methods of the present invention, thelymphocytes are selected from the group consisting of T cells, B cells,natural killer (NK) cells, and a combination thereof.

In one embodiment of the methods of the present invention, thelymphocytes are T cells.

In another embodiment of the methods of the present invention, the Tcells produce IFN-γ.

In one embodiment of the methods of the present invention, the severeand/or rapidly progressing fibrosis is pulmonary fibrosis.

In one embodiment of the methods of the present invention, the severeand/or rapidly progressing fibrosis is associated with a conditionselected from the group consisting of scleroderma lung disease,saracoidosis, Wegener's granulomatosis, infections, asbestosis, ionizingradiation exposure, lupus, rheumatoid arthritis, hypersensitivitypneumonitis, nonspecific interstitial pneumonitis, Hamman-Rich Syndrome,diffuse fibrosing alveolitis, idiopathic pulmonary fibrosis, andcombinations thereof.

In one embodiment of the methods of the present invention, the severeand/or rapidly progressing fibrosis is further mediated bymetalloproteinases including, but not limited to MMP2, MMP9, or acombination thereof.

In one embodiment, the severe or progressing pulmonary fibrosis isassociated with tissue injury.

In another embodiment of the methods of the present invention, thetissue injury is caused by an injury-inducing agent selected from thegroup consisting of anticonvulsant drug, antipsychotic drug,antidepressant drug, anti-inflammatory drug, anti metabolic drug,antimicrobial drug, biologic response modifiers, cardiovascular drug,chemotherapeutic drug, immunosuppressive drug, and combinations thereof.

In one embodiment of the methods of the present invention, theantimicrobial drug is selected from the group consisting ofnitrofurantoin, sulfasalazine, tetracycline, minocycline, sulfonamides,parpa-aminosalicyclic acid, ethambutol, ampicillin, cephalosporin, andcombinations thereof.

In one embodiment of the methods of the present invention, thecardiovascular drug is selected from the group consisting of amiodarone,angiotensin-converting enzyme (ACE) inhibitor, and combinations thereof.

In one embodiment of the methods of the present invention, thechemotherapeutic drug is selected from the group consisting ofbleomycin, mitomycin-C, busulfan, cyclophosphamide, nitrosourea,procarbazine, melphalan, paclitaxel, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts hematoxylin and eosin (H&E) staining of lung sectionsafter intratracheal instillation of AdV-CCL18 (A,B), bleomycin (C,D), ortheir combination (E,F). Instillation of AdV-NULL caused minimalinfiltration of inflammatory cells on days 3-7 that completely resolvedby day 14. Instillation of AdV-CCL18 and subsequent CCL18 overexpressionmanifested in peribronchial and perivascular lymphocytic infiltrationaround small (A) and large (B) bronchi and vessels (arrows), and minimalinterstitial lymphocytic infiltration (arrowheads). Instillation ofbleomycin resulted in the characteristic diffuse insterstitialalveolitis accompanied by distortion of alveolar architecture (C,D). Thecombined effect of CCL18 overexpression and bleomycin injury manifestedin large areas of distorted alveolar architecture and more pronouncedinfiltration throughout the lung (E,F), and more severe interstitiallymphocytic infiltration remotely from bronchi and vessels (arrows).Immunohistochemical staining for CD4+ cells (G-I) confirmed theperibronchial and perivascular nature of the infiltration in the CCL18overexpression model (G), more scattered presence of T cells in the lungparenchyma in the bleomycin injury model (H), and the combinedlymphocytic accumulation pattern (adjacent to anatomical structures plusinterstitial) upon combined CCL18 overexpression and bleomycin injury tothe lungs (I).

FIG. 2 depicts absolute (A) and relative (B) BAL cell count, and totalprotein concentration in lung homogenates. A. The majority of BAL cellswere represented by macrophages and lymphocytes in all groups, withadditive effects of CCL18 overexpression and bleomycin on total cell andlymphocyte counts (BLM 0.03 U/mouse shown). B: Stacked column plotshowing relative macrophage (top, open bars) and lymphocyte (bottom,shaded bars) content in bronchoalveolar lavage, mean percent±SD of totalBAL cells (the averaging procedure may lead to a combine cell countsslightly exceeding 100%). Other cell types were represented byneutrophils and epithelial cells and did not jointly exceed 3% in any ofthe groups; there was not difference in the neutrophil content betweenthese groups (p>0.05). As previously reported (11), mice instilled witheither PBS or AdVNULL (Ctrl) showed no difference (p>0.05). The secondinstillation was with either PBS or BLM as shown. The differencesbetween Ctrl and CCL18 are significant in all cases (p<0.05, Student'st-test, three to eight animals per group, repeated on three differentoccasions with consistent results). These data suggest that CCL18overexpression and bleomycin injury have additive effect on lymphocyticaccumulation in the lungs. C: Levels of total protein in lunghomogenates in the combined CCL18 overexpression and bleomycin injurygroup exceeded those in any other group (p<0.05), further suggestingthat these two factors facilitate pulmonary inflammation in the additivefashion.

FIG. 3 depicts total hydroxyproline per left lung (Hyp) as a surrogatemeasure of collagen content. A: 4 Mean Hyp, tag±SD, three to eightanimals per group The second instillation was with either PBS or BLM asshown. Notice that in the absence of bleomycin, CCL18 overexpressionstimulated collagen accumulation (p<0.05). In contrast, CCL18overexpression partially neutralized the effect of bleomycin on collagenaccumulation (p<0.05 for both doses of bleomycin). The expected additiveeffect of CCL18 overexpression and bleomycin injury on collagenaccumulation is shown with the unfilled bars/dashed lines. B: AverageHyp level per lung in six independent experiments as described in A forBLM 0.03 U; standard deviations were similar to those shown in A. Eachpoint indicates average Hyp, presented as percent of Hyp value incorresponding PBS-treated controls, three to eight animals per group.The connecting lines represent six independent experiments performed onseparate occasions.

FIG. 4 depicts matrix metalloproteinases (MMP) in the lungs of miceoverexpressing CCL18 and/or treated with bleomycin. A,B: ELISA of lunghomogenates for total (pro- and active) MMP2 (Panel A) and pro-MMP9(Panel B). Data are shown as mean pg/pg total protein±SD, eight totwelve animals per group, repeated on two different occasions withsimilar results. Overexpression of CCL18 and injury with bleomycin actadditively on accumulation of matrix metalloproteinases. In Panel A, thedifferences between CCL18-expressing and non-expressing animals weresignificant (p<0.05) in each bleomycin dose group. Similar tendencieswere observed in Panel B although the differences betweenCCL18-expressing and non-expressing animals did not reach significance(p>0.05) within each bleomycin dose group, due to low concentration ofpro-MMP9 and low signal-to-noise ratio. C: Immunohistochemistry for MMP9(pro- and total), ×200 magnification, showing additive effect of CCL18overexpression and bleomycin exposure on accumulation of MMP9-producingcells in the lung (brown staining). No staining was detected withisotype control antibody (not shown) D: Zymogram of lung homogenates.Sample loading was normalized to total protein (Bio-Rad assays).Molecular weight markers, kDa, are indicated on the right. Expectedlocations of pro-MMP9 (92 kDa), active MMP9 (82 kDa), pro-MMP2 (72 kDa),and active MMP2 (62 kDa) are indicated with arrows. Repeated on threeseparate occasions in different groups of animals, with similar results.

FIG. 5 depicts ELISA of lung homogenates for IFN-γ(A), TNF-α(B), MCP-1(C), and active TGF-β (D), pg/ml, mean±SD. Data averaged from three toeight animals per group in (A,B,C) and eight to twelve animals per groupin (D). The increases in IFN-γ (A), TNF-α (B), and MCP-1 (C) in theCCL18+BLM group were significant (p<0.05) in comparison with any othergroup and appeared additive of the effects of CCL18 alone and BLM alone.In (D), the level of active TGF-β in the CCL18+BLM group was different(p<0.05, Student's t-test) from any other group, but not additive of theeffects of CCL18 alone and BLM alone. In these experiments, 0.03 U BLMwas utilized.

FIG. 6 depicts changes in the total levels of hydroxyproline, foldincrease versus control, in the lung of mice overexpressing CCL18 andchallenged with high dose of bleomycin, upon treatment with an MMPinhibitor GM6001 or neutralizing anti-MMP9 antibody. Both treatmentssignificantly abrogated accumulation of hydroxyproline and cytokinelevels where indicated with asterisks (p<0.05, Student's t-test, threeto eight animals per group). Treatment with GM6001 in the group of miceinstilled with 0.03 U of bleomycin alone did not attenuate the levels ofhydroxyproline (207.3±18.8 pg/lung vs 222.7±15.6 pg/lung, non-treated vstreated groups, respectively, p>0.05).

FIG. 7 is shows the antifibrotic effect produced by increasing CCL18expression in the lungs, and the advantages of increasing CCL18 toprevent or treat pulmonary fibrosis. The claimed method of increasingCCL18 is compared to the method of injecting recombinant antifibroticcytokines to reduce pulmonary fibrosis. The injection of recombinantanti fibrotic cytokines failed to reduce pulmonary fibrosis because thehalf-life of the injected recombinant cytokine is short, and itsbioavailability in the pulmonary tissues is minimal. In contrast, theincrease in CCL18 expression produced by the present invention causes anunexpected enhancement in the observed levels of antifibrotic factors,e.g., by T lymphocytic infiltration and/or production of IFN-γ andTNF-α, into the lungs. The attracted T cells become an important localsource of antifibrotic cytokines, and is advantageous because theinfiltration of T cells allow for: 1) the continuous production ofantifibrotic cytokines that, in comparison to the method of injectingcytokines, is independent of the amount of application and half-life ofthe cytokine, and 2) local, tissue specific production of antifibroticfactors which, in comparison to the method of injecting cytokines,ensure for substantial bioavailability of the protective antifibroticcytokines in the tissues. Because the claimed method provides for alocal source of T cells, the present invention also circumvents thesystemic side effects and problems associated with crossing theendothelial barrier that is encountered by the injection of cytokines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of treating severe or rapidlyprogressing pulmonary fibrosis in a subject in need of a treatmentthereof. The methods comprise increasing the activity of pulmonary andactivation-regulated chemokine (CCL18) in the lungs of the subject,whereby increasing CCL18 activity modulates the activity of at least oneantifibrotic factor in the lungs of the subject.

CCL18 is a profibrotic “CC chemokine” that is chemotactic for T cellsthat is constitutively expressed in the lungs. CCL18 has a high aminoacid sequence identity with macrophage inflammatory protein-1 alpha(MIP-1a), but does not bind to the MIP-1a receptors CCR5 and CCR1.Monocyte chemotactic protein-1 (MCP-1) is the only other known CCchemokine capable of increasing collagen production in fibroblasts.CCL18 promotes fibrosis and is expressed at high levels in the lungs,particularly by activated lung macrophages, although other tissuemacrophages and dendritic cells may secrete CCL18.

In addition to its profibrotic activity, CCL18 attracts naive andactivated CD4+ and CD8+T cells. Fibrosis was observed in animalsinfected with a replication-deficient adenovirus harboring the CCL18gene. The levels of CCL18 produced in adenoviral models may, however, besufficient to attract T-cells, which may, in turn, be contributing tocollagen accumulation and fibrosis observed in the adenovirus-infectedanimals.

CCL18 is a cytokine that is differentially regulated in classically andalternatively activated macrophages. For example, interferon-7 inhibitsCCL18 production in activated macrophages, whereas interleukin 4 (IL-4),IL-13, and IL-10 induce CCL18 production. Furthermore, the developmentof pulmonary fibrosis is generally associated with predominantexpression of type 2 cytokines in the lungs, thus type 2 cytokines notonly promote lung fibrosis by acting directly on lung, fibroblasts, butalso indirectly through alternative pathway to increase production ofCCL18.

CCL18 increases the phosphorylation of extracellular signal-regulatedkinase (ERK), a kinase involved in a variety of second messenger cellsignaling cascades, in a time-dependent manner. In addition,pharmacological inhibition of ERK blocked the CCL18-induced stimulationof collagen production in fibroblast. Accordingly, CCL18 directlystimulates collagen production in lung and dermal fibroblasts byactivating intracellular signaling through the ERK pathway.

CCL18 directly stimulates type I collagen production in at least lungand dermal fibroblasts. This increase in collagen mRNA indicates thateither an increase in gene transcription or an increase in mRNAstability may be responsible for the increased collagen production inresponse to CCL18. It is possible that CCL18 may also affect theintracellular pools of free proline, thus accounting for, at least inpart, CCL18's stimulation of collagen production in fibroblasts.

For the purposes of the present invention, “CCL18” includes full lengthCCL18 protein, as well as functional fragments thereof. Using standardassays to measure for typical CCL18 activity, one of skill in the artcould assay for functional fragments of CCL18. The fragments need not beas active or effective as full length, provided that the CCL18 fragmentsare derived from the full-length CCL18 and that the fragments have atleast one activity associated with the full-length CCL18 protein.

Activated alveolar macrophages are an abundant source of CCL18, and lungmacrophages are actively involved in lung inflammation involved inpulmonary fibrosis. Studies have shown that CCL18 is in elevatedconcentrations in BAL fluids taken from patients with scleroderma lungdisease.

The present inventors have recently reported that mice infected with areplication-deficient adenovirus encoding CCL18 but not with a similarcontrol virus develop selective T lymphocytic infiltration of the lungs,as well as moderate transient T cell-dependent collagen accumulation(11). Phenotypic characterization of the infiltrating cells incomparison with normally present pulmonary T cells revealed minimal, ifany, activation, including lack of elevated expression of severalprofibrotic factors (1). However, the lymphocytic infiltration coincidedwith the sites of accumulation of active TGF-131 and collagen (11),suggesting that the infiltrating T cells directly contributed to theprofibrotic effect of CCL18. In support of this notion, systemicdepletion of T cells completely abrogated lymphocytic infiltration andcollagen accumulation in CCL18-overexpressing mice (11).

In patients with lung fibrosis, increases in pulmonary levels of CCL18occur in association with lung inflammation and fibrosis (1-7). The lunginflammation is characterized by an influx of lymphocytes, collagenaccumulation, and increase in proinflammatory factors such aschemokines, macrophage inflammatory protein 4 (MIP-4), alternativemacrophage activation-associated CC chemokine 1 (AMAC-1), dendriticcell-derived chemokine 1 (DCCK1), and small secreted cytokine A 18(SCYA-18).

As used herein, the “activity of CCL18” is used to mean the expressionof CCL18 or the actions or effects of CCL18. Thus, increasing theactivity of CCL18 within a cell would, for the purposes of the presentinvention, include increasing the expression of CCL18. Similarly,increasing the stability of mRNA that codes for CCL18 would alsoconstitute increasing the activity of CCL18. Of course, increasing theactivity of CCL18 would also include administering the CCL18 peptidedirectly or indirectly to the target cells. In one embodiment, themethods of increasing the activity of CCL18 include, but are not limitedto, increasing the expression of CCL18 in a cell or population of cellsby delivering one or more non-native nucleic acids to a target cell thatcodes for CCL18 or a peptide with CCL18 activity. The nucleic aciddelivery into target cell can be accomplished using standardtransfection techniques, which include, but are not limited to,magnetofection, cationic lipid-based delivery, or electroporation. Thenucleic acid delivery may be via a viral or non-viral vector mediateddelivery into the target cells. As used herein, a “non-native” nucleicacid is intended to mean a nucleic acid that the target cell does notnormally contain. Thus, a “non-native nucleic acid” includes, but is notlimited to, an extra copy of a nucleic acid that codes for CCL18, evenif the nucleic acid is wild-type to the target cell. Of course, a“non-native nucleic acid” also includes, but is not limited to, nucleicacids that are heterologous to the target cell.

The nucleic acid constructs can be any construct capable of deliveringnon-native nucleic acids to target cells, and the invention is notlimited to or dependent upon a particular type of construct for deliveryof the nucleic acid encoding for CCL18. Examples of vectors and plasmidsare abundant in the art and commercially available. In one embodiment,viral vector are used to increase the expression of CCL18 and include,but are not limited to, replication-deficient recombinant adenoviralvectors and replication-deficient recombinant adeno-associated viralvectors.

In accordance with the present invention, the “target cell” may include,but is not limited to, pulmonary cells, epithelial cells, cerebralcells, breast cells, myocardial cells, musculoskeletal cells, livercells, neuronal cells, vascular cells, vein cells, skin cells, pancreascells, spleen cells, gall bladder cells, kidney cells, urogenital cells,ocular cells, or other cells susceptible to fibrosis, provided that thecell produces or causes a response to the increased activity of CCL18.

In accordance with the present invention, a “CCL18-responsive cell” is acell in which an increase in expressed CCL18 can trigger a biologicalresponse either in vitro or in vivo. Such cells may include, but are notlimited to, T cells, B cells, dendritic cell chemokine (DC-CK1),hematopoietic progenitor cells, fibroblasts, monocytes, macrophages, ora combination thereof. For instance, CCL18 may trigger T cell productionof cytokines such as IFN-γ, or activate monocytes or macrophages toproduce cytokines such as T helper cell type 2 (Th2)-related cytokines(e.g., IL-4, IL-10, or IL-13), or glucocorticoids (GC).

The present invention relates to methods of treating severe or rapidlyprogressing pulmonary fibrosis. As used herein, the term “treatment” isused to indicate a procedure which is designed ameliorate one or morecauses, symptoms, or untoward effects of an abnormal condition in asubject. Likewise, the term “treat” is used to indicate performing atreatment. The treatment can, but need not, cure the subject, i.e.,remove the cause(s), or remove entirely the symptom(s) and/or untowardeffect(s) of the abnormal condition in the subject. Thus, a treatmentmay include treating a subject to attenuate symptoms such as, but notlimited to, discomfort, pain, shortness of breath (particularly withexertion), chronic dry, hacking, cough, fatigue and weakness, discomfortin the chest, loss of appetite and rapid weight loss, in a subject, ormay include removing or decreasing the severity of the root cause of theabnormal condition in the subject. Treatment also includes treatingafter-arising symptoms that are related to the initiation pulmonaryfibrosis.

As used herein, the term “subject” is used interchangeably with the term“patient,” and is used to mean an animal, in particular a mammal, andeven more particularly a non-human or human primate.

The term “fibrosis” is used herein as it is in the medical arts andrefers to the formation or development of fibrous connective tissue inan organ or tissue due to collagen and/or other connective tissueaccumulation. A molecule that promotes fibrosis is one that directly orindirectly contributes to the accumulation of collagenous and/or otherconnective tissue.

The term “severe and/or rapidly progressing fibrosis” is used to referto significant overgrowth, scarring or hardening that occurs throughouta particular organ or tissue, e.g., the lungs, and indicates an abnormalcondition in a subject that is marked by excessive accumulation ofcollagenous and/or other connective tissue in comparison to a normalcondition in which the fibrous tissue is a normal constituent of anorgan or tissue. The abnormal condition causes the formation ordevelopment of fibrous connective tissue from excessive collagen and/orother connective tissue accumulation in an organ or tissue as a resultof a reactive process, in contrast to a formation of fibrous tissue thatis a normal constituent of an organ or tissue. Examples of pathologicand excessive fibrotic accumulation include, but are not limited to,pulmonary fibrosis, benign prostate hypertrophy, fibrocystic breastdisease, uterine fibroids, ovarian cysts, endometriosis, coronaryinfarcts, cerebral infarcts, myocardial fibrosis, musculoskeletalfibrosis, post-surgical adhesions, liver fibrosis, cirrhosis, realfibrotic disease, or fibrotic vascular disease, e.g., atherosclerosis,varix, or varicose veins, scleroderma, Alzheimer's disease, diabeticretinopathy and glaucoma.

Severe and/or rapidly progressing fibrosis may be determined based on anincrease in collagen accumulation over normal individuals, as clinicallyjudged by lower pulmonary functions measures, including, but not limitedto, diffusing capacity for carbon monoxide (DLCO) and forced vitalcapacity (FVC). The increase in collagen and/or other connective tissueaccumulation over normal individuals may be evaluated according to knownmethods for measuring tissue collagen accumulation and usinghistological analyses evaluating changes in pulmonary architecture andcollagen-specific staining, including, but not limited to, trichromestaining. The extent of collagen and/or other connective tissueaccumulation in severe and/or rapidly progressing fibrosis over normalindividuals may, in turn, be determined by using standard methods knownin the art to measure the molecular and cellular changes in cells andtissues that are related to fibrogenesis. Severe and/or rapidlyprogressing fibrosis may be determined based on, for example, acombination of clinical changes (including, but not limited to, dyspnea,cough, bibasilar crackles), pulmonary function changes (including, butnot limited to, worse DLCO and FVC values), and radiographic andhistological changes consistent with interstitial pneumonia. Inparallel, the diagnosis may be confirmed by measuring the levels ofbiomarkers in the lungs that characterize the pathogenesis of fibrosisincluding, but not limited to, profibrotic growth factors, reactiveoxygen species (ROS), cell signaling factors, and proinflammatorycytokines. Profibrotic growth factors include, but are not limited to,transforming growth factor-beta (TGF-β), connective tissue growth factor(CTGF), or a combination thereof. For example, TGF-β is believed to be akey mediator of tissue fibrosis as a consequence of extracellular matrix(ECM) accumulation in pathologic states such as scleroderma. TGF-β isknown to induce the expression of ECM proteins in mesenchymal cells, andto stimulate the production of protease inhibitors that preventenzymatic breakdown of the ECM. CTGF, which is induced by TGF-β, hasbeen reported to mediate stimulatory actions of TGF-β ECM synthesis.Furthermore, severe and/or rapidly progressing fibrosis may bedetermined using standard methods known in the art to measure levels ofantifibrotic factors such as IFN-γ and/or TNF-α.

In one embodiment, the severity of pulmonary fibrosis is evaluated usinga standard hydroxyproline assay. It is well-known that hydroxyproline isincorporated into collagen, thus levels of hydroxyproline can bedirectly correlated to levels of collagen from a sample. Furthermore, itis also known that fibrosis may not be uniform throughout an affectedorgan, and that the fibrosis may be “patchy.” Thus, for the purposes ofthe present invention, “moderate fibrosis” is intended to mean an organor tissue, for example the lung, where at least a portion of the organor tissue has greater than a 1 to about a 1.5 fold increase in thelevels of hydroxyproline over levels of hydroxyproline in the organ ortissue in subjects that do not have fibrosis. “Severe firbrosis” isintended to mean an organ or tissue, for example the lung, where atleast a portion of the organ or tissue has greater than about a 1.5increase in the levels of hydroxyproline over levels of hydroxyprolinein the organ or tissue in subjects that do not have fibrosis. It iswell-known to those of ordinary skill in the art that other means ofmeasuring and determining fibrosis are known in the art with such othermeans of measuring and determining fibrosis representing a furtherembodiment of the present invention.

As used herein, “about” refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited value) that one wouldconsider equivalent to the recited value (e.g., having the same functionor result). In some instances, the term “about” may include numericalvalues that are rounded to the nearest significant figure.

In one embodiment of the present invention, the fibrosis that is treatedor prevented from progressing by the methods described herein ispulmonary fibrosis. In more particular embodiments, the pulmonaryfibrosis is a symptom of a condition including, but not limited to,scleroderma lung disease, saracoidosis, Wegener's granulomatosis,infections, asbestosis, ionizing radiation exposure, lupus, rheumatoidarthritis, hypersensitivity pneumonitis, nonspecific interstitialpneumonitis, Hamman-Rich Syndrome, diffuse fibrosing alveolitis,idiopathic pulmonary fibrosis, or a combination thereof.

The severe or rapidly progressing fibrosis ay also be associated withtissue injury. As used herein, the term “tissue injury” is used to meandamage or harm caused to the structure or function of a tissue by anagent that may be physical or chemical. The injury induced by an agentto a target cell in the present invention may cause a variety ofsymptoms, in particular, fibrosis and inflammation. The agent of thepresent invention may also cause injury by exacerbating an underlyingdisease in a predisposed subject, or cause the disease.

In one embodiment, the injury could be cellular injury. The injury maybe mediated by at least one immune cell selected from monocytes,macrophages, lymphocytes, plasma cells, and a combination thereof. Inparticular, the injury by an injury-inducing agent of the presentinvention is to a pulmonary cell resulting in pulmonary injury thatcauses pulmonary fibrosis and/or pulmonary inflammation.

As used herein, “cellular injury” to a target cell may be reversible orirreversible. Cellular injury includes, but is not limited to, cellularswelling (cellular hypertrophy), cellular atrophy (cell shrinkage),fatty change (cells fail to metabolize fatty acids and accumulatelipids), or a combination thereof. Additional examples of cellularinjury include, but are not limited to, changes in the density of themitochondrial matrix, cell membrane disruption, nuclear shrinkage(pyknosis), nuclear dissolution (karyolysis), nuclear break up(karyorrhexis), lysosome rupture, apoptosis, cellular necrosis, cellularhyperplasia (an increase in the number of cells which may have increasedcellular volume caused by physiological stress or pathological stimuli),or a combination thereof.

The structure and function of a cell are interdependent. An injuriousagent may target a particular aspect of a cell structure or function andlead to cellular injury. Mechanisms of cellular injury include, but arenot limited to, cell membrane damage (e.g., complement-mediated lysisvia the membrane attack complex, bacterial toxins, free radicals),mitochondrial damage leading to inadequate aerobic respiration (e.g.,hypoxia, cyanosis), ribosomal damage leading to altered proteinsynthesis (e.g., alcohol, antibiotics), increased production of reactiveoxygen or nitrogen species, and nuclear damage (e.g., viruses,radiation, free radicals). Potential causes of cellular injury include,but are not limited to, hypoxia, immunological, infection bymicroorganisms, genetic, physical, and chemical, such a drug-relatedfibrosis.

The pulmonary injury induced by at least one injury-inducing agentincludes, but is not limited to, pulmonary fibrosis and pulmonaryinflammation. The pulmonary injury may be mediated by at least oneimmune cell selected from the group consisting of monocytes,macrophages, lymphocytes, plasma cells, and a combination thereof.

The agent that may be used to induce pulmonary injury includes, but isnot limited to, anticonvulsant drug, anti-inflammatory drug,antimetabolic drug, antimicrobial drug, biologic response modifiers,cardiovascular drug, chemotherapeutic drug, immunosuppressive drug,illicit drug, or a combination thereof.

The anticonvulsant drug may include, but is not limited to,carbamazepine, chlordiazepoxide, fluoxetine, phenothiazines, phenytoin,trazodone, tricyclics, or a combination thereof.

The anti-inflammatory drug may include, but is not limited to, aspirin,gold, methotrexate, penicillamine, or a combination thereof.

The antimetabolic drug may include, but is not limited to, azathioprine,cytarabine, fludarabine, gemcitabine, 6-mercaptopurine, methotrexate, ora combination thereof.

The antimicrobial drug may include, but is not limited to,nitrofurantoin, sulfasalazine, tetracycline, minocycline, sulfonamides,parpa-aminosalicyclic acid, ethambutol, ampicillin, cephalosporin, or acombination thereof.

The biologic response modifiers may include, but are not limited to,granulocyte-macrophage colony-stimulating factor, interferon,interleukin-2, tumor necrosis factor, or a combination thereof.

The cardiovascular drug may include, but is not limited to, amiodarone,angiotensin-converting enzyme (ACE) inhibitor, or a combination thereof.

The chemotherapeutic drug may include, but is not limited to, bleomycin,mitomycin-C, busulfan, cyclophosphamide, nitrosourea, procarbazine,melphalan, paclitaxel, or a combination thereof.

The immunosuppressive drug may include, but is not limited to,cyclosporine, corticosteroids, azathioprine, cyclophosphamide, or acombination thereof.

The illicit drug may include, but is not limited to, cocaine, heroin,methadone, methylphenidate, narcotic, sedative, or a combinationthereof.

The treatment methods of the present invention are used to modulate theactivity of at least one anti-fibrotic factor in the lungs of a subject.As used herein, the term “modulate” means to vary, alter, or change. Forinstance, the injury-inducing agent of the present invention modulatesfibrosis by increasing antifibrotic or decreasing profibrotic factorsthat play a role in the formation or development of excess fibrousconnective tissue in an organ or tissue.

As used herein, the term “antifibrotic” means regression of fibrosis.Alternatively, the term “profibrotic” means progression of fibrosis.“Regression” as used herein means a lessening of symptoms or reductionof the accumulation of collagenous and/or other connective tissue or thereduction of the total amount of excessive collagen in a particulartissue, relating to the fibrotic condition before employing the methodsof the present invention without complete disappearance of the symptoms.“Progression” as used herein means advancement or progressing ofsymptoms.

The antifibrotic factors include, but are not limited to,metalloproteinases, interleukins, interferons, cytokines, chemokines,chemotactic molecules, macrophages, lymphocytes, or a combinationthereof. In particular, the antifibrotic factors include but are notlimited to matrix metalloproteinase-2 (MMP2), MMP9, tumor necrosisfactor alpha (TNF-α), T cells, interferon gamma (IFN-γ), andcombinations thereof. Other antifibrotic factors are well-known in theart and need not be repeated herein.

In one embodiment, the methods comprise administering to a subject aneffective amount of a compound or molecule that increases the activityof CCL18. As used herein, the term “effective amount” means an amountthat is sufficient to achieve the stated or desired effect, and can be asimple matter of titration. An effective amount of CCL18 used in themethods of the present invention is an amount sufficient to increase theactivity, e.g., concentrations, of CCL18 in a target cell in comparisonto an untreated (control) cell.

An effective amount of the drug, as the term is used herein, refers tothat amount of drug which is effective therapeutically in the desiredtreatment. In accordance with the present invention, the effectiveamount of an agent used in the present methods is one that elicits anyone or all of the effects often associated with the in vivo biologicalactivity of the agent. In addition, the effective amount of an agent inthe present methods may also include one that elicits an in vitrobiological effect by agent.

For instance, in accordance with the present invention, an effectiveamount of CCL18 may be below 1 ng/ml to initiate attraction of T cellsto the lungs. Alternatively, at the concentrations above 1 ng/ml, up to300 ng/ml or 1000 ng/ml or more, an effective amount used in the methodsis likely to act not only to attract T cells to the lungs, but alsodirectly to affect pulmonary fibroblasts. These concentrations refer tothe average concentrations measured by standard techniques such as ELISAor Western blotting in lung homogenates or alternatively, to localconcentrations in the immediate vicinity of cells such as T cells orfibroblasts.

A medicament useful for the methods of treating, preventing orpreventing the progression of fibrosis may be prepared by standardpharmaceutical techniques known in the art, depending upon the mode ofadministration and the particular disease to be treated. The medicamentwill usually be supplied as part of a sterile, pharmaceuticalcomposition which will normally include a pharmaceutically acceptablecarrier. This pharmaceutical composition may be in any suitable form,(depending upon the desired method of administering it to a subject). Itmay be provided in unit dosage form, will generally be provided in asealed container and may be provided as part of a kit, which may includeinstructions for use and/or a plurality of unit dosage forms.

Dosages of the substance of the present invention can vary between widelimits, depending upon the disease or disorder to be treated, the ageand condition of the individual to be treated, etc. and a physician willultimately determine appropriate dosages to be used.

The pharmaceutical composition may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such compositions maybe prepared by any method known in the art of pharmacy, for example byadmixing the active ingredient with the carrier(s) or excipient(s) understerile conditions.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; as powders orgranules; as solutions, syrups or suspensions (in aqueous or non-aqueousliquids; or as edible foams or whips; or as emulsions). Suitableexcipients for tablets or hard gelatine capsules include lactose, maizestarch or derivatives thereof stearic acid or salts thereof. Suitableexcipients for use with soft gelatine capsules include for examplevegetable oils, waxes, fats, semi-solid, or liquid polyols etc. For thepreparation of solutions and syrups, excipients which may be usedinclude for example water, polyols and sugars. For the preparation ofsuspensions oils (e.g. vegetable oils) may be used to provideoil-in-water or water in oil suspensions. In certain situations, delayedrelease preparations may be advantageous and compositions which candeliver, for example, AET or a derivative thereof in a delayed orcontrolled release manner may also be prepared. Prolonged gastricresidence brings with it the problem of degradation by the enzymespresent in the stomach and so enteric-coated capsules may also beprepared by standard techniques in the art where the active substancefor release lower down in the gastro-intestinal tract.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research,3(6):318 (1986).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils. When formulated in anointment, the active ingredient may be employed with either a paraffinicor a water-miscible ointment base. Alternatively, the active ingredientmay be formulated in a cream with an oil-in-water cream base or awater-in-oil base. Pharmaceutical compositions adapted for topicaladministration to the eye include eye drops wherein the activeingredient is dissolved or suspended in a suitable carrier, especiallyan aqueous solvent. Pharmaceutical compositions adapted for topicaladministration in the mouth include lozenges, pastilles and mouthwashes. Pharmaceutical compositions adapted for rectal administrationmay be presented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable compositions wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalationinclude fine particle dusts or mists which may be generated by means ofvarious types of metered dose pressurized aerosols, nebulizers orinsufflators.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solution which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation substantially isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Excipients which may beused for injectable solutions include water, alcohols, polyols,glycerine and vegetable oils, for example. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carried, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets. The pharmaceutical compositions maycontain preserving agents, solubilising agents, stabilising agents,wetting agents, emulsifiers, sweeteners, colourants, odourants, salts(substances of the present invention may themselves be provided in theform of a pharmaceutically acceptable salt), buffers, coating agents orantioxidants. They may also contain therapeutically active agents inaddition to the substance of the present invention.

The present invention also relates to methods of screening a compoundthat may alter the progression of severe or rapidly progressingpulmonary fibrosis. The screening methods comprise administering aninjury-inducing agent to a control and test population of cells, whereinthe injury-inducing agent is known to produce severe or rapidlyprogressing pulmonary fibrosis. A test procedure is also administered tothe test population of injured cells. In one specific embodiment, thetest procedure is known or suspected of being able to increase theactivity of pulmonary and activation-regulated chemokine (CCL18) in cellpopulations. In response to the test procedure, data is observed orgathered wherein the data comprises test level activities of CCL18activity. In one specific embodiment, levels of CCL18 are determinedusing the activity of at least one antifibrotic factor in or from thetest population of cells. The test data are compared to control data,wherein the control data comprises standard activity levels of CCL18and/or the activity levels of the at least one antifibrotic factors thatare established in the control population of injured cells. A differencein test activity levels and standard activity levels indicates that thetest procedure may be capable of altering the progression of severe orrapidly progressing pulmonary fibrosis.

The test procedures that are used in the screening methods can be anyprocedure that is known or suspected of being able to increase theactivity of CCL18 in a target cell population. Such test proceduresinclude, but are not limited to, increasing expression or biologicalhalf-life of proteins that stimulate or are believed to stimulate CCL18production. Other test procedures include, but are not limited to,administering compounds or compositions to the test cells that canincrease expression, increase the stability of mRNA or increase thehalf-life of the CCL18 peptide. Once a relationship is establishedbetween the test procedure and CCL18, regardless of the setting, thetest procedure is a candidate for use in the present invention.

Data is collected or gathered towards at least one antifibrotic factoras described herein and the test data are compared to a standard. Thestandard may be any set point, provided the set point can be compared tothe test data. For example, the standard may be the levels concentrationor activity of a particular antifirotic factor in a normal cell ortissue, such as a lung. Alternatively, the standard may be the levels,concentration or activity of a particular antifirotic factor in aslightly fibrotic cell culture or tissue. Or the standard may be thelevels/concentration or activity of a particular antifirotic factor in aseverely fibrotic cell or tissue. The setting of the screening methodsmay be any setting where data may be gathered such as, but not limitedto, a cell culture setting, a tissue mount or a whole animal, etc. Thestandard activity levels may or may not be known prior to thecommencement of the screening methods, and may established at any time.

INDUSTRIAL APPLICABILITY

The claimed invention provides unexpected and superior results that areadvantageous over previous therapies and methods of treating and/orpreventing fibrosis, e.g., pulmonary fibrosis. The claimed methods allowfor: 1) continuous infiltration of T cell infiltration and infiltrationof antifibrotic factors (e.g., IFN-γ), 2) bioavailability ofantifibrotic factors in the lungs due to localization of antifibroticfactors in the lung tissues, 3) minimal side effects related to systemicadministration of antifibrotic factors. In embodiments where theactivity of CCL18 is not increased systemically, problems related tocrossing the endothelial barrier are circumvented.

For instance, in the past, antifibrotic cytokines (e.g. IFN-γ) have beentherapeutically administered by systemic injection, but failed to reducepulmonary fibrosis because the half-life of the injected recombinantcytokine is short, and its local bioavailability in mammalian (e.g.,human) tissues (e.g., the lungs) is minimal, likely due to theendothelial barrier. In contrast, the present invention allows for anelevated and continuous T-cell lymphocytic infiltration as a result ofthe increased CCL18 activity, e.g., expression, in the inflammatorymilieu of the lung, i.e., increased CCL18 expression causes a continuouselevation of antifibrotic cytokines such as IFN-γ and TNF-α. Becauseincreasing the activity of CCL18 by the claimed method may not involvesystemic injections of antifibrotic cytokines, the present invention maycircumvent the systemic side effects and minimal bioavailabilityassociated with injecting cytokines (e.g., crossing the endothelialbarrier). Thus, the T cells that infiltrate because of the delivery ofCCL18 become an important local source of antifibrotic cytokines, andimportantly, such source is 1) continuous and independent of injectionsand the half-life of the cytokine, 2) local and tissue-specific, and 3)ensures substantial bioavailability of protective cytokines in thetissues (because there is no need for the cytokines to cross theendothelial barrier). FIGS. 7A and 7B summarizes the advantageous andunexpected antifibrotic effects of increasing CCL18 expression by theclaimed method.

EXAMPLES Example 1 Experimental Animal Models

Ten- to twelve-week-old C57BL/6 female mice weighing 18-21 g werepurchased from The Jackson Laboratory (Bar Harbor, Me.) and maintainedin sterile microisolator cages with sterile rodent feed and acidifiedwater. Daily maintenance of mice was performed in the Baltimore Va.Medical Center Research Animal Facility that is approved by theAssociation for Assessment and Accreditation of Laboratory Animal Care(AAALAC). The animals were treated in accordance with a researchprotocol that has been approved by the University of MarylandInstitutional Animal Care and Use Committee (IACUC). Mice were weigheddaily using a calibrated scale.

Recombinant adenoviral vectors AdV-CCL18 and AdV-NULL were constructed,validated, and used as previously reported (11). Intratrachealinstillation of 50 μl of bleomycin solution (Sigma-Aldrich, St. Louis,Mo.) or PBS was performed in a fashion similar to instillation ofadenoviruses (11).

To address the possibility that the effects of CCL18 on collagenproduction in vivo may depend on the presence and severity of theinflammatory milieu, intratracheal instillation of AdV-CCL18 or, ascontrols, of AdV-NULL or PBS was performed (day 0). Selected animalswere sacrificed to confirm the expected CCL18 expression dynamics aspreviously reported (see FIG. 1 in ref. 11). At the peak of CCL18production (day 7), intratracheal instillation of bleomycin wasperformed to induce lung inflammation and fibrosis; alternatively, PBSwas instilled as control.

As a result of these two instillations, four groups of mice were formed,as described below. The control group (Ctrl) was instilled first withAdV-NULL or PBS and then with PBS again. It has been previously reportedthat mice instilled with AdV-NULL or PBS were not phenotypically orhistologically different beyond three days following instillation (11).The CCL18 alone group overexpressed CCL18 as a result of AdV-CCL18instillation. The second instillation in this group was with PBS. Thebleomycin alone (BLM) group received the first instillation of eitherAdV-NULL or PBS, and the second instillation of either 0.01 U or 0.03 Uof bleomycin.

These doses of bleomycin were selected as insufficient to achieve aplateauing effect on lung inflammation and fibrosis, based on theresults of preliminary experiments in which titrated doses (0.005 U-0.1U per mouse) were instilled. Finally, the combined CCL18 overexpressionand bleomycin injury group (CCL18+BLM) received AdV-CCL18 (firstinstillation) and bleomycin (second instillation). On day 21 followingthe first instillation (day 14 after the second instillation), mice wereeuthanized by 002 asphyxiation followed by cervical dislocation. ELISAassays of lung homogenates were used to confirm the expected decline ofCCL18 levels by day 21 following AdV-CCL18 instillation (11); suchdecline was independent of the nature of the agent used for the secondinstillation (bleomycin or PBS).

For depletion of lymphocytes, some of the animals were first injectedwith antilymphocyte serum (ALS; Accurate, Westbury, N.Y.), on days −4,−2, and 0 relevant to the first intratracheal instillation, and thedecrease in the amount of lymphocytes to <5% of the initial levels wasconfirmed by flow cytometry, as described (11). In some other cases,mice were treated with a broad-spectrum MMP inhibitor GM6001 (Chemicon,Temecula, Calif.) intraperitoneally at 2 mg/mouse daily for the last 5days before euthanasia; or with anti-MMP9 neutralizing antibody(Calbiochem, San Diego, Calif.) intraperitoneally at 60 fag/mouse ondays 14 and 18 after first instillation.

Example 2 Histological Examination of the Lungs, Bronchoalveolar Lavage,and Flow Cytometry

Immediately postmortem, the lungs were rapidly dissected free ofextraneous tissues and filled with either formalin-free fixative(Anatech, Battle Creek, Mich.) for subsequent hematoxylin and eosinstaining or with 1:1 mixture of PBS and TissueTek OCT compound (Sakura,Torrance, Calif.) for subsequent immunohistochemical analyses. Cryostatsections, stainings, controls, and image analyses were performed asdescribed (11).

For BAL, the animals were euthanized, and lung lavage was performedimmediately postmortem through an 18-gauge blunt-end needle secured inthe trachea as described (11). Differential cell count in BAL sampleswere performed after staining of cytospin preparations with a ProtocolHema 3 staining set (Fisher, Kalamazoo, Mich.) by at least twotechnicians who were blinded to the identity of the samples. The flowcytometric analyses of BAL cells were performed after staining withdirectly labeled antibodies (BD PharMingen, San Diego, Calif.) orcorresponding isotype controls as described (11).

Example 3 Determination of Hydroxyproline Content ELISA Analyses, andZymographic Analyses of Lung Homogenates

Pulmonary levels of hydroxyproline were measured as surrogate of totalcollagen, as described (11). Briefly, the snap-frozen lungs were crushedunder liquid nitrogen, thawed in 0.5 ml of PBS containing a proteaseinhibitor cocktail (Sigma), and further homogenized in a glasshomogenizer. The solid tissue was separated by centrifugation; thesupernatant was diluted two fold with the ELISA sample buffer, and usedfor ELISA analyses of total and active TGF-β, IL13, TNF-α, IFN-γ, MCP-1(CCL2), total (pro- and active) MMP-2, and pro-MMP-9 (all kits purchasedfrom R&D Systems, Minneapolis, Minn.).

Total protein was measured using Bio-Rad assay (Hercules, Calif.). Thesolid tissue was hydrolyzed in 5N NaOH at 120° C. for 30 minutes in anautoclave. The mixture was then reacted with chloramine T and Ehrlich'sreagent to produce a chromophore, which was quantified byspectrophotometry at 550 nm. A second aliquot of the original lunghomogenate was used for colorimetric detection and quantification fortotal protein content using Bio-Rad assay.

For zymographic analyses, lungs were homogenized in 50 mM Iris-NCIbuffer containing 1 mM monothioglycerol, and the solid tissue wasseparated by centrifugation. The supernatants were normalized for totalprotein and loaded onto Novex® 10% zymogram gels containing 0.1% gelatin(Invitrogen, Carlsbad, Calif.). After electrophoretic separation gelswere renatured, developed at 37° C. overnight, and stained withColloidal Blue stain (Invitrogen) following manufacturer'srecommendations.

Example 4 In Vitro Chemotaxis Assays

Chemotaxis assays were used as described in (11) to determine whetherCCL18 selectively attracts regulatory T cells. Briefly, human T cellspurified from PBMC as described in (11) were seeded in triplicates inthe upper chamber using Costar Transwell inserts (3-pm pore size;Costar, Cambridge, Mass.) an incubated, with or without rhCCL18 in thelower chamber, at 37° C. for 4 hours. The cells that migrated into thelower chamber, as well as the cells that remained in the upper chamber,were analyzed for expression of cell surface CD4, CD25, andintracellular FoxP3 by flow cytometry. Data are reported as the mean±SD.Differences between groups were evaluated with Student's 2-tailedunequal variance t-test and Mann-Whitney U test P values less than 0.05were considered statistically significant.

Example 5 Pulmonary Responses to Adenoviral Delivery of CCL18 andInstillation of Bleomycin

To evaluate the combined effect of CCL18 overexpression and bleomycininjury on the lungs, each animal in EXAMPLE 1 received two intratrachealinstillations. The first instillation of AdVCCL18 or AdV-NULL or PBS,was followed by the second instillation of bleomycin or PBS, asdescribed in EXAMPLE 1.

Mice instilled intratracheally with AdV-CCL18, AdV-NULL, or PBS showedno signs of morbidity such as body weight loss (p>0.05, one-way ANOVA),ruffled fur, dehydration, diarrhea, hunched posture, or decreased motoractivity at any time postinfection. Mice instilled with bleomycin showedan expected total body weight loss of maximum 6.2±2.7% followinginstillation of 0.01 U of bleomycin and maximum 10.1±3.2% followinginstillation of 0.03 U of bleomycin. This statistically significant(p<0.05 by one-way ANOVA, data not shown) weight loss was observed ondays 5-20 following the second intratracheal instillation of bleomycinbut not PBS, independent of the nature of the agent used for the firstinstillation (AdV-CCL18, AdV-NULL or PBS). There was minimalpost-operational mortality that was not significantly different betweenanimal groups. Thus, bleomycin injury to the lung caused more severemorbidity that manifested in weight loss than pulmonary CCL18overexpression.

Histologically, instillation of AdV-CCL18 but not AdV-NULL or PBS causedperivascular and peribronchial lymphocytic infiltration (FIG. 1A,B). TheT lymphocytic nature of these cells was confirmed by immunohistochemicalstaining of lung sections for CD3, CD4, and CD8. Instillation ofbleomycin caused the characteristic diffuse interstitial fibrosingalveolitis (FIG. 1C,D).

The combined effect of CCL18 overexpression and bleomycin injuryexceeded the effect of each factor alone. It manifested in greaterdestruction of alveolar architecture, more pronounced perivascular andperibronchial infiltration, and massive lymphocytic accumulation notonly in the peribronchial and perivascular areas, but also in theinterstitium (FIG. 1E,F). Immunohistochemical analyses for CD3, CD4,CD8, and TCR confirmed such infiltration patterns (an example for CD4+cells is shown in FIG. 1G-I).

Overexpression of CCL18 and bleomycin injury had an additive effect on Tcell content in BAL and total protein concentration in lung homogenates(FIG. 2). Flowcytometric analyses of BAL cells revealed increases in Tlymphocytes in CCL18 overexpressing mice as previously reported (seeFIG. 3 in ref. 11). Consistent with previous reports (11), gating onCD3+CD4+ and CD3+CD8+ cells revealed the following CD41CD8 ratios:0.97±0.17 in CCL18 overexpressing mice, 1.61±0.22 in mice challengedwith bleomycin, and 0.98±0.09 in mice challenged with CCL18overexpression and bleomycin injury (n=10 in each group).

In all cases, more than 90% of lung cells were immunohistochemicallynegative for proliferating cell nuclear antigen (PCNA), suggesting thatthey are not dividing cells and that the observed dynamics of theinfiltration is due to trafficking to the lungs (not shown).Immunohistochemically and flowcytometrically, there was minimal (<1%)presence of CD19+ or B220+ cells. These observations suggested thatbleomycin injury and overexpression of CCL18 together elicit a moresevere proinflammatory effect on the lung than each of these factorsalone, particularly manifesting in additive accumulation of Tlymphocytes in the lungs.

Some rare regulatory T cells (FoxP3+) were present in the infiltratesimmunohistochemically. Flowcytometric analyses of the permeabilized BALT cells revealed that 8.94±1.04% of CD4+CD25+FoxP3+ were present inCCL18 overexpressing mice, whereas 8.48±1.22% of such cells were presentin mice overexpressing CCL18 in combination with bleomycin injury (n=5in each group, p>0.05). Separate in vitro chemotaxis experiments usingTranswell® system were performed to separate CCL18-responding (lowerchamber) from nonresponding (upper chamber) human T cells freshlypurified from the PBMC population.

Flow cytometry analyses revealed that in both populations of T cells,those that did or did not respond chemotactically to CCL18, thefractions of CD4+CD25+FoxP3+ cells were similar (6.95±1.5% and7.08±1.3%, respectively, p>0.05). These in vivo data from animal modelsand in vitro data obtained with human purified T cells suggest thatCCL18, alone or in combination with bleomycin, is not a selectiveattractor of regulatory T cells.

Example 6 Collagen Production and Accumulation in Pulmonary FibrosisModels

Experiments tested whether CCL18 overexpression and bleomycin incombination would enhance pulmonary fibrosis. In contrast to theadditive effect of CCL18 overexpression and injury with bleomycin on thelevels of T lymphocytes accumulating in the lungs, the CCL18overexpression had a partially neutralizing effect on bleomycin-inducedcollagen accumulation (FIG. 3).

Consistent with previous observations (11), overexpression of CCL18 byitself caused a moderate increase in total pulmonary hydroxyprolinecontent, compared to the increases caused by bleomycin alone (FIG. 3A).However, the combined effect of CCL18 overexpression and bleomycininjury [black bars in the BLM groups in FIG. 3A] was below the expectedadditive effect of these two factors (unfilled bars, dashed outlines inFIG. 3A). Moreover, the combined effect of CCL18 overexpression andbleomycin injury on collagen accumulation [black bars in the BLM groupsin FIG. 3A] was significantly lower than the effect of bleomycin injuryalone (grey bars). This observation was made in six independentexperiments, with consistent results (FIG. 3B).

An average decrease in total lung Hyp was 33.4±3.9%, based on sixindependent experiments shown in FIG. 3B. Thus, CCL18 overexpressioncaused T cell-dependent (11) mild pulmonary fibrosis in an otherwisehealthy lung (11), yet it partially protected against severe fibroticinjury caused by bleomycin (FIG. 3). This effect was observed atdifferent non-saturating concentrations of bleomycin (FIG. 3A) and atdifferent times, on day 21 as shown in FIG. 3, and on day 28.

Example 7 Determination of the Mechanisms for the Paradoxical Regulationof Collagen Levels in the Combined CCL18 Overexpression and BleomycinInjury Model

In considering the possible mechanisms that may be involved in theunexpected regulation of collagen levels in the lungs in the combinedCCL18 overexpression and bleomycin injury model, the present inventorsfocused on well known regulators of connective tissue homeostasis,metalloproteinases MMP-2 and MMP-9 (reviewed in 29-32) and majorcytokines known to be involved in regulation of inflammation andfibrosis TGF-0, IL-13, TNF-α, IFN-γ, MCP-1 (CCL2) (reviewed in 33) aspossible regulators of the observed dynamics in collagen levels.

Levels of MMP2 and MMP9 are generally elevated in fibrotic lungdiseases, these metalloproteinases are known to contribute both pro- andantifibrotically (29-32). The ELISA assays showed an additive effect ofCCL18 overexpression on the levels of pro- and active MMP2 and pro-MMP9in the lung homogenates (FIG. 4A,B). This additive effect was confirmedimmunohistochemically for total (pro- and active) MMP9 (FIG. 4C).Zymographic analyses also revealed an increase in gelatinase activity inthe combined CCL18 overexpression and bleomycin injury group (FIG. 4D).

The levels of total TGF-β and IL-13 measured by ELISA variedinsignificantly between the groups (p>0.5, Student's t-test, data notshown), similar to our previous report (11). However, overexpression ofCCL18 and bleomycin injury additively upregulated the pulmonary levelsof IFN-γ, TNF-α, and MCP-1 (FIG. 5A). Mediators IFN-γ and TNF-α havebeen shown to have both pro- and antifibrotic effects in vivo, with theprofibrotic effects being secondary to inflammation, whereas these twofactors in vitro are potent inhibitors of collagen production (reviewedin 33).

Thus, the increased levels of these cytokines provide a possibleexplanation for the decrease in collagen accumulation in the combinedCCL18 overexpression and bleomycin injury model. Reciprocally, thelevels of a known profibrotic regulator, active TGF-β, in the combinedinjury model were lower than in mice subjected to bleomycin injury alone(FIG. 5D), which mirrored the changes in hydroxyproline (see FIG. 3).

These observations suggested that multiple mediators, metalloproteinasesand cytokines, are involved in the observed regulation of collagenlevels in the combined CCL18 overexpression and bleomycin injury model.

Example 8 Effect of Lymphocyte Depletion and Camp Neutralization onCollagen Accumulation in the Lungs

It was Determined if CCL18 by itself was sufficient to cause the changesin collagen accumulation, or whether there is a need for pulmonary Tcells in mediating the observed effects.

Real-time PCR and ELISA showed that mice treated with anti-lymphocyteserum (ALS) before the administration of AdV-CCL18 did not affect CCL18expression in the lung. However, there was complete abrogation of theperivascular and peribronchial infiltration and collagen accumulationconsistent with previous reports (11). In addition, consistent withother previous reports (22,23,25), treatment with ALS did not have asignificant effect on collagen accumulation in the bleomycin injurymodel (p>0.05, Student's t-test).

In the combined CCL18 overexpression and bleomycin injury model treatedwith ALS, pulmonary levels of collagen did not differ from the bleomycininjury alone treated with ALS (205.8±14.1 pg/lung vs 199±12.3 pg/lung,respectively, p>0.05, Student's t-test, data not shown). Thus, theeffect of bleomycin injury alone on collagen accumulation appears to beT cell-independent, whereas depletion of T cells eliminates the effectof CCL18 overexpression on collagen accumulation the normal orbleomycin-damaged lung.

The contribution of MMPs to tissue fibrosis is complex (29-32). Thechanges in the levels of MMPs (see FIG. 4) were determined in regard tothe regulation of collagen and cytokine levels. Administration ofGM6001, a broad-spectrum pharmacological MMP inhibitor, significantlyabrogated pulmonary levels of hydroxyproline, active TGF-β, TNF-α andIFN-γ in the combined injury model (FIG. 6), suggesting a centralinvolvement of MMPs in inflammatory and fibrotic processes.Administration of neutralizing anti-MMP9 antibody had no effect on thelevels of active TGF-β or TNF-α but abrogated the levels of IFN-γ andfurther abrogated the levels of hydroxyproline, suggesting that MMP2 islikely to be involved in regulation of TGF-β and TNF-αlevels. Thus, MMP2and MMP9 contribute to the regulation of collagen and cytokine levels inthe combined injury model.

Example 9 Recombinant Adenovirus-Mediated CCL18 Gene Delivery andBleomycin-Induced Drug Injury

Adenovirus-mediated gene delivery combined with bleomycin-induced injuryto the lung is conducted according to established methods (22,34,35).

Mice are instilled with AdV-CCL18(11), and then at the peak of CCL18production (day 7), a second intratracheal instillation is performedwith either PBS (as a control) or bleomycin, in lower (0.01 U/mouse) orhigher (0.03 U/mouse) dose to induce lung inflammation and fibrosis. Theselected doses of bleomycin alone were found to be insufficient toachieve a plateauing effect on lung inflammation and fibrosis inpreliminary experiments.

The bleomycin challenge but not instillation of PBS caused astatistically significant transient loss of body weight on days 5-20following the second intratracheal instillation, independent of thenature of the agent used for the first instillation (AdV-CCL8, AdV-NULL,or PBS). Thus, bleomycin injury to the lung causes more severe morbiditymanifested in weight loss than pulmonary CCL18 overexpression. Thecombined effect of CCL18 overexpression and bleomycin injury exceededthe effect of each factor alone on the severity of histological changes.It manifested in greater destruction of alveolar architecture, morepronounced perivascular and peribronchial infiltration, and massivelymphocytic accumulation not only in the peribronchial and perivascularareas, but also in the interstitium (see FIG. 1).

Overexpression of CCL18 and the bleomycin injury had an additive effecton T cell content in the BAL samples and total protein concentration inlung homogenates (see FIG. 2). Thus, overexpression of CCL18 and thebleomycin injury together elicit a more severe proinflammatory effect onthe lung than each of these factors alone.

In contrast to the additive effect on inflammation, CCL18 overexpressionunexpectedly attenuated the severe bleomycin-induced collagenaccumulation (see FIG. 3). This finding suggested that althoughCCL18-induced T lymphocytic infiltration is by itself mildly profibroticto a healthy lung (11), the very same infiltration may be partiallyprotective against severe fibrosis in a proinflammatory profibroticsetting in the lungs.

Although a consistently reproducible phenomenon (see FIG. 3B), theamplitude of decline in pulmonary collagen level in the combined modelcompared with bleomycin alone is constitutes about 33.4±3.9% of thenormal collagen content. However, it is important to consider thatfibrosis correlates with decline in lung function (36), and that thepresent method provided an approximately 30% increase in pulmonaryfunction in comparison to another therapy that reportedly resulted inonly 3% higher forced vital capacity than placebo and which iscelebrated as a significant achievement in treating patients withscleroderma lung disease (37).

The present invention shows that further enhancing the antifibroticregulation in the lungs may be accomplished by therapeuticallymanipulating the local pulmonary milieu and/or the phenotypes ofinfiltrating T lymphocytes.

There are sporadic data on antifibrotic effects of T lymphocytes invitro (38,39) and in vivo (40). The idiopathic pulmonary fibrosis (IPF)patients with a higher percentage of BAL T lymphocytes may be moreresponsive to treatment (41). In combination with the present findings,these data show that T cell infiltration in patients with pulmonaryfibrosis is part of an antifibrotic feedback loop, suggesting thateliminating T cells from the inflamed lung may promote fibrosis and thushave a counterintuitive deleterious effect.

Depletion of lymphocytes with an anti-lymphocyte serum abrogated theperivascular and peribronchial infiltration and collagen accumulation(not shown), consistent with our previous report (11). The presentinventors have found that systemic antibody-mediated depletion of Tlymphocytes completely abrogates the effects of CCL18 overexpression inthe lungs (11).

Similarly, treatment with antilymphocyte serum in the combined CCL18overexpression and bleomycin injury model did abrogate the partialprotective effect of CCL18 overexpression in the combined model inimmunocompetent mice; pulmonary levels of collagen in the Tlymphocyte-depleted animals that overexpressed CCL18 and were challengedwith bleomycin did not differ from the immunocompetent mice challengedwith bleomycin alone.

Accordingly, T lymphocytes may be therapeutically modulated to actantifibrotically, instead of being targeted and eliminated from thelungs.

The present inventors have investigated the mechanism of the paradoxicalregulation of the collagen accumulation in the combined CCL18overexpression and bleomycin injury model. One possible mechanism mayinvolve regulatory T cell; however, no differences between the studiedgroups of animals were found in the content of CD4+CD25+FoxP3+ cells.Also, in vitro chemotaxis assays revealed that CCL18 did not selectivelyattract such regulatory T cells. Together, these observations suggestedthat T regulatory T cells explain the differences in collagenaccumulation between animal groups.

Metalloproteinases MMP-2 and MMP-9 are well known regulators ofconnective tissue homeostasis that are involved in lung inflammation andfibrosis and act dually, proteolytically and non-proteolytically, in acomplex concentration-dependent fashion (29-32). Depending on thespecifics of the inflammatory milieu and local concentration of MMPs,their effects may be either pro- or antifibrotic.

These data show that overexpression of CCL18 and injury with bleomycinacted additively on MMP2 and MMP9 levels and activity in the lungs. Aspart of addressing the observed mechanism, the levels of pro- andantifibrotic cytokines were measured. The levels of proinflammatorycytokines TNF-α and IFN-γ were additively upregulated by CCL18overexpression and the bleomycin injury (see FIG. 5A). These two factorsare known to have complex effect on tissue fibrosis, including theirdirect and indirect regulation of collagen production by fibroblasts(reviewed in 33).

One line of evidence, in in vitro studies suggests that they are potentdirect inhibitors of collagen production in fibroblasts. In contrast, insome animal models, each of these cytokines indirectly facilitatedfibrosis through inflammation-dependent mechanisms (reviewed in 33). Theresults on the elevation of these potentially antifibrotic cytokinesshown in FIGS. 5 and 6 are consistent with their antifibrotic action,leading to decline in collagen levels in the combined injury model (seeFIG. 3).

Reciprocally, the levels of active TGF-β declined in the combined injurymodel (see FIG. 5B), also consistent with the changes in collagenaccumulation (see FIG. 3). Thus, the levels of the profibrotic factor,active TGF-β, mirrored those of collagen, whereas the levels ofpotentially antifibrotic factors TNF-α and IFN-γ changed reciprocally,in agreement with the attenuation of the collagen content in thecombined CCL18 overexpression and bleomycin injury model.

Therefore, numerous factors are involved in the observed paradoxicalregulation of collagen accumulation in the combined CCL18 overexpressionand bleomycin injury model. Although TGF-β is a central mediator ofbleomycin-induced lung fibrosis, increases in the total levels of thispowerful profibrotic cytokine are difficult to detect because of theoverall high basal level of inactive TGF-β (e.g. see FIG. 5C in ref.42). Significant changes in total pulmonary TGF-β in the studied modelscould not be detected. The present inventors also found no significantchanges in the levels of a potent profibrotic cytokine IL-13.

Neutralization of MMPs with a broad-spectrum inhibitor GM6001 furtherattenuated the decline in collagen accumulation and the levels of theprofibrotic and proinflammatory cytokines in animals with the combinedCCL18 overexpression and bleomycin injury (see FIG. 6), confirming asignificant role for MMPs and a potential for therapeutic modulation ofthese enzymes in lung fibrosis. A selective neutralization of MMP9 witha specific neutralizing antibody attenuated the levels of collagen andIFN-7 but not TNF-α and active TGF-61, indicating that MMP2 may berequired for regulation of levels of the two latter cytokines.

The novelty of these results is that the CCL18-mediated T lymphocyticinfiltration is profibrotic in the otherwise healthy lungs but it isunexpectedly partially antifibrotic when superimposed on a secondprofibrotic injury (bleomycin). It also appears that the regulation ofCCL18-mediated pulmonary inflammation and fibrosis is complex and occursthrough mechanisms that involve T lymphocytic infiltration, matrixmetalloproteinases, and profibrotic and proinflammatory cytokines. Thesimplistic approach to developing new antifibrotic therapies in whichpulmonary T lymphocytes are selectively targeted to diminish the degreeof fibrosis should be reconsidered, as these cells play a partiallyprotective role and could be phenotypically modulated to act even moreantifibrotically.

LIST OF REFERENCES

The following references are incorporated by reference in theirentirety:

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What is claimed is:

1. A method of treating severe or rapidly progressing pulmonary fibrosisin a subject in need of a treatment thereof, the method comprisingincreasing the activity of pulmonary and activation-regulated chemokine(CCL18) in the lungs of the subject, whereby increasing CCL18 activitymodulates the activity of at least one antifibrotic factor in the lungsof the subject.
 2. The method of claim 1, wherein increasing theactivity comprises increasing the expression of a peptide with CCL18activity.
 3. The method of claim 2, wherein the peptide is CCL18.
 4. Themethod of claim 3, wherein the CCL18 is native to the subject.
 5. Themethod of claim 4, wherein the CCL18 is not native to the subject. 6.The method of claim 3, wherein increasing the expression of CCL18comprises transfection.
 7. The method of claim 6, wherein transfectioncomprises a viral vector or a non-viral vector.
 8. The method of claim6, wherein the transfection comprises a technique selected from thegroup consisting of magnetotransfection, cationic lipid-based delivery,electroporation and combinations thereof.
 9. The method of claim 1,wherein the severe or rapidly progressing pulmonary fibrosis isassociated with a disorder selected from the group consisting ofscleroderma lung disease, saracoidosis, Wegener's granulomatosis,infections, asbestosis, ionizing radiation exposure, lupus, rheumatoidarthritis, hypersensitivity pneumonitis, nonspecific interstitialpneumonitis, Hamman-Rich Syndrome, diffuse fibrosing alveolitis,idiopathic pulmonary fibrosis, idiopathic pulmonary fibrosis andcombinations thereof.
 10. The method of claim 1, wherein the at leastone antifibrotic factor in the lungs of the subject is selected from thegroup consisting of matrix metalloproteinase-2 (MMP2), matrixmetalloproteinase-9 (MMP9), tumor necrosis factor alpha (TNF-α),interleukin-8 (TL-8), interleukin-1 (IL-1), T cells, B cells, naturalkiller (NK) cells, interferon gamma (IFN-γ), interferon alpha (IFN-α),and combinations thereof.
 11. The method of claim 1, wherein the severeor rapidly progressing pulmonary fibrosis is associated with tissueinjury.
 12. The method of claim 11, wherein the tissue injury is causedby an injury-inducing agent selected from the group consisting of ananticonvulsant drug, an antipsychotic drug, an antidepressant drug, ananti-inflammatory drug, an antimetabolic drug, an antimicrobial drug,biologic response modifiers, a cardiovascular drug, a chemotherapeuticdrug, an immunosuppressive drug, and combinations thereof.
 13. Themethod of claim 12, wherein the antimicrobial drug is selected from thegroup consisting of nitrofurantoin, sulfasalazine, tetracycline,minocycline, sulfonamides, parpa-aminosalicyclic acid, ethambutol,ampicillin, cephalosporin, and a combination thereof.
 14. The method ofclaim 12, wherein the cardiovascular drug is selected from the groupconsisting of amiodarone, angiotensin-converting enzyme (ACE) inhibitor,and a combination thereof.
 15. The method of claim 12, wherein thechemotherapeutic drug is selected from the group consisting ofbleomycin, mitomycin-C, busulfan, cyclophosphamide, nitrosourea,procarbazine, melphalan, paclitaxel, and a combination thereof.
 16. Themethod of claim 15, wherein the chemotherapeutic drug is bleomycin. 17.A method of treating severe or rapidly progressing pulmonary fibrosis ina subject in need of a treatment thereof, the method comprisingadministering to the subject a means for increasing the activity ofpulmonary and activation-regulated chemokine (CCL18) in the lungs of thesubject, whereby increasing CCL18 activity modulates the activity of atleast one anti fibrotic factor in the lungs of the subject.
 18. A methodof screening a compound that may alter the progression of severe orrapidly progressing pulmonary fibrosis, the method comprising a)administering an injury-inducing agent to a control and test populationof cells, wherein the injury-inducing agent is known to produce severeor rapidly progressing pulmonary fibrosis, and b) administering a testprocedure to the test population of injured cells, c) observing testlevel activities of pulmonary and activation-regulated chemokine (CCL18)d) comparing the test activity levels of CCL18 with a standard activitylevel of CCL18, wherein the standard activity level of CCL18 areestablished in the control population of injured cells, wherein anincrease in the activity levels of CCL18 in the test population over thestandard CCL18 activity levels indicates that the test procedure may becapable of altering the progression of severe or rapidly progressingpulmonary fibrosis.
 19. The method of claim 18, wherein the testprocedure is suspected of being able to increase the activity ofpulmonary and activation-regulated chemokine (CCL18) in cellpopulations.
 20. The method of claim 18, wherein the activity levels ofCCL18 are measured using the activity of at least one antifibroticfactor.